The recently recognized family of patatin-like proteins is widely distributed among bacteria. They are defined by the presence of a patatin domain, which encodes phospholipase A2 activity. Although the vast majority of these proteins remain to be characterized, most of those examined to date are secreted by type III, type IV, or type V secretion systems, are translocated into host cells, and have attributes of virulence factors. Once in the intracellular environment, these phospholipases cause disruption of normal host cell physiology or cell death. The best characterized representative of the patatin-like family of proteins is ExoU of Pseudomonas aeruginosa. This protein, which is associated with virulence in both animal models and human infections, is secreted by the P. aeruginosa type III secretion pathway and causes rapid lysis of a broad spectrum of eukaryotic cells by a phospholipase A2 dependent mechanism. The overall goal of this application is to build upon prior work by our laboratory and others to further characterize the molecular mechanisms of ExoU and to extend these findings to other patatin-like proteins. We hypothesize that the membrane localization domain of ExoU targets this effector protein to the plasma membrane by binding phosphatidylinositol-4,5-bisphosphate and that this binding markedly enhances ExoU phospholipase A2 activity. We hypothesize that ExoU undergoes multimerization once at the plasma membrane. Finally, we suggest a therapeutic strategy whereby the cytotoxic activity of ExoU can be exploited to eradicate P. aeruginosa and other bacteria that target lytic toxins to neutrophils. In this application, we propose aims to test each of these hypotheses. The completion of these aims will further define the molecular mechanisms of patatin-like proteins and lay the foundation for the development of novel therapeutic interventions for patients infected by bacteria that lyse neutrophils.